1. Field of the Invention
This invention is directed, in part, to polymers and gels comprising a polypeptide and a polysaccharide, and to methods of making the same.
2. Description of Related Art
Hydrogels are providing new opportunities for a variety of medical applications. Biomaterials Science: An Introduction to Materials in Medicine (Ratner B D et al., eds.; 1996); Okano T. Biorelated Polymers and Gels (1998). Examples include the use of hydrogels as skin substitutes, adhesives, matrices for drug delivery, and scaffolds for tissue engineering. See, e.g., Biomaterials Science: An Introduction to Materials in Medicine; Peppas N A and Sahlin J J, Biomaterials 17:1553–1561 (1996); McCulloch I and Shalaby S W, “Tailored polymeric materials for controlled delivery systems” (Washington D.C.: American Chemical Society, 1998); Dinh S M, DeNuzzio J D and Comfort A R, “Intelligent materials for controlled release” (Washington D.C.: American Chemical Society, 1999); Mallapragada S, Tracy M, Narasimhan B, Mathiowitz E and Korsmeyer R., “Biomaterials for drug delivery and tissue engineering” (Warrendale, Pa.: Materials Research Society, 2001); Lee K Y and Mooney D J, Chem. Rev. 101:1869–1879 (2001). In many of these applications it would be desirable if the hydrogel could be formed in situ. For instance, it would be possible to “implant” materials using minimally invasive methods if systems were available that could be injected as solutions and gelled only after injection. Elisseeff J, et al., Proc. Natl. Acad. Sci. 96:3104–3107 (1999). Further, in situ gel formation would allow gels to be created that filled the available space. Gutowska A, Jeong B and Jasionowski M., Anat Rec. 263:342–349 (2001); Gerentes P, et al., Biomaterials 23:1295–1302 (2002). Obviously, major constraints on such gel-forming systems are that they must be non-toxic and biocompatible.
There are a few common approaches for creating gels that could be extended to in situ systems. One approach commonly used for in vitro gel formation is to initiate polymerization reactions in the presence of multi-functional monomers. Since these multi-functional monomers are incorporated into two (or more) growing polymer chains the reaction leads to a three-dimensional network. Huang Y, Szleifer I and Peppas N A, Macromolecules 35:1373–1380 (2002). An example of this approach for in situ applications is the cyanoacrylate adhesives. Smith D C, “Adhesives and sealants,” Biomaterials Science: An Introduction to Materials in Medicine p. 319–328 (Ratner B D et al., eds.; 1996). Since low molecular weight and reactive monomers are used, this approach raises concerns of toxicity and compatibility. A second approach for forming gels which is particularly attractive for in situ applications is to use “smart” polymers that gel in response to the conditions experienced after injection/application. Galaev I Y and Mattiasson B, Trends Biotechnol 17:335–340 (1999). Typical smart polymers respond to changes in temperature or pH and can be made of natural (e.g., gelatin) or synthetic (e.g., poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide))polymers. Bromberg L E and Barr D P, Macromolecules 32:3649–3657 (1999); Huibers P D T, et al., Macromolecules 32:4889–4894 (1999). This approach is attractive for in situ applications although there are currently few smart polymers that are also biocompatible. A third approach for gel formation is to initiate the crosslinking of soluble, linear polymers or macromonomers. Typically, crosslinking is initiated using light or low molecular weight crosslinking agents such as glutaraldehyde. See, e.g., Elisseeff J, et al., J. Biomed. Mater. Res. 51:164–171 (2000); Ono K, et al., J. Biomed. Mater. Res. 49:289–295 (2000); Bryant S J and Anseth K S, Biomaterials 22:619–626 (2001); Behravesh E, Jo S, Zygourakis K and Mikos A G, Biomacromolecules 3:374–381 (2002); Temenoff J S, et al., J. Biomed. Mater. Res. 59:429–437 (2002); Koh W G, Revzin A and Pishko M V, Langmuir 18:2459–2462 (2002); Mi F-L, et al., Carbohydrate Polymers 41:389–396 (2000); Bigi A, et al., Biomaterials 22:763–768 (2001). For in situ applications there are safety concerns associated with the use of such low molecular weight and reactive compounds (i.e., monomers or initiators).
In many cases, natural polymers are advocated as biomaterials because they may be non-toxic, biodegradable, and have low immunogenicities. Yannas I V, “Natural Materials,” Biomaterials Science: An Introduction to Materials in Medicine p. 84–94 (Ratner B D et al., eds.; 1996). In addition to reducing or avoiding adverse effects, biopolymers may actually offer beneficial properties. For instance collagen is a major component of the extracellular matrix of tissue, and collagen (or gelatin) based materials are reported to promote cell attachment and growth. Koide M, et al., J. Biomed. Mater. Res. 27:79–87 (1993); Stanton J S, et al., J. Mater. Sci.-Mater. Med. 6:739–744 (1995). Chitosan has also been reported to have antimicrobial hemostatic, and wound healing properties that could be exploited for biomaterials. Muzzarelli R, et al., Antimicrob. Agents Chemother. 34:2019–2023 (1990); Mi F L, et al., Biomaterials 22:165–173 (2001); Mi F, et al., J. Biomed. Mater. Res. 59:438–449 (2002); Rao S B and Sharma C P, J. Biomed. Mater. Res. 34:21–28 (1997); Ishihara M, et al., Biomaterials 23:833–840 (2002); Muzzarelli R, et al., Biomaterials 9:247–252 (1988); Ueno H, et al., Biomaterials 20:1407–1414 (1999); Cho Y W, et al., Biomaterials 20:2139–2145 (1999).
Apart from having the desirable chemical and biological properties, biomaterials must have the mechanical properties (e.g., strength, hardness and durability) required by whatever applications they are used in. Anseth K, et al., Biomaterials 17:1647–1657 (1996). The mechanical properties of tissue are often conferred by protein-polysaccharide conjugates (e.g., proteoglycans and mucins), and there has been considerable recent interest in generating such conjugates for various applications, especially as dressings and scaffolds for tissue engineering. See, e.g., Yannas I V, et al., Proc. Natl. Acad. Sci. USA 86:933–937 (1989); Yannas I V and Burke J F, J. Biomed. Mater. Res. 14:65–81 (1980); Choi Y S, et al., J. Biomed. Mater. Res. 48:631–639 (1999); Angele P, et al., Tissue Eng. 5:545–554 (1999). Unfortunately, the complexity of protein-polysaccharide conjugates has made it difficult to recover or synthesize these glycoconjugates.